Gene editing provides a tool to investigate genes involved in reproduction of pigs.

CRISPR-Cas9 gene editing technology provides a method to generate loss-of-function studies to investigate, in vivo, the specific role of specific genes in regulation of reproduction. With proper design and selection of guide RNAs (gRNA) designed to specifically target genes, CRISPR-Cas9 gene editing allows investigation of factors proposed to regulate biological pathways involved with establishment and maintenance of pregnancy. The advantages and disadvantages of using the current gene editing technology in a large farm species is discussed. CRISPR-Cas9 gene editing of porcine conceptuses has generated new perspectives for the regulation of endometrial function during the establishment of pregnancy. The delicate orchestration of conceptus factors facilitates an endometrial proinflammatory response while regulating maternal immune cell migration and expansion at the implantation site is essential for establishment and maintenance of pregnancy. Recent developments and use of endometrial epithelial "organoids" to study endometrial function in vitro provides a future method to screen and target specific endometrial genes as an alternative to generating a gene edited animal model. With continuing improvements in gene editing technology, future researchers will be able to design studies to enhance our knowledge of mechanisms essential for early development and survival of the conceptus.

Chloride channel accessory 1 gene deficiency causes selective loss of mucus production in a new pig model.

Morbidity and mortality of respiratory diseases are linked to airway obstruction by mucus but there are still no specific, safe, and effective drugs to correct this phenotype. The need for better treatment requires a new understanding of the basis for mucus production. In that regard, studies of human airway epithelial cells in primary culture show that a mucin granule constituent known as chloride channel accessory 1 (CLCA1) is required for inducible expression of the inflammatory mucin MUC5AC in response to potent type 2 cytokines. However, it remained uncertain whether CLCLA1 is necessary for mucus production in vivo. Conventional approaches to functional biology using targeted gene knockout were difficult due to the functional redundancy of additional Clca genes in mice not found in humans. We reasoned that CLCA1 function might be better addressed in pigs that maintain the same four-member CLCA gene locus and the corresponding mucosal and submucosal populations of mucous cells found in humans. Here we develop to our knowledge the first CLCA1-gene-deficient (CLCA1-/-) pig and show that these animals exhibit loss of MUC5AC+ mucous cells throughout the airway mucosa of the lung without affecting comparable cells in the tracheal mucosa or MUC5B+ mucous cells in submucosal glands. Similarly, CLCA1-/- pigs exhibit loss of MUC5AC+ mucous cells in the intestinal mucosa without affecting MUC2+ mucous cells. These data establish CLCA1 function for controlling MUC5AC expression as a marker of mucus production and provide a new animal model to study mucus production at respiratory and intestinal sites.

Cardiovascular Development and Congenital Heart Disease Modeling in the Pig.

Background Modeling cardiovascular diseases in mice has provided invaluable insights into the cause of congenital heart disease. However, the small size of the mouse heart has precluded translational studies. Given current high-efficiency gene editing, congenital heart disease modeling in other species is possible. The pig is advantageous given its cardiac anatomy, physiology, and size are similar to human infants. We profiled pig cardiovascular development and generated genetically edited pigs with congenital heart defects. Methods and Results Pig conceptuses and fetuses were collected spanning 7 stages (day 20 to birth at day 115), with at least 3 embryos analyzed per stage. A combination of magnetic resonance imaging and 3-dimensional histological reconstructions with episcopic confocal microscopy were conducted. Gross dissections were performed in late-stage or term fetuses by using sequential segmental analysis of the atrial, ventricular, and arterial segments. At day 20, the heart has looped, forming a common atria and ventricle and an undivided outflow tract. Cardiac morphogenesis progressed rapidly, with atrial and outflow septation evident by day 26 and ventricular septation completed by day 30. The outflow and atrioventricular cushions seen at day 20 undergo remodeling to form mature valves, a process continuing beyond day 42. Genetically edited pigs generated with mutation in chromatin modifier SAP130 exhibited tricuspid dysplasia, with tricuspid atresia associated with early embryonic lethality. Conclusions The major events in pig cardiac morphogenesis are largely complete by day 30. The developmental profile is similar to human and mouse, indicating gene edited pigs may provide new opportunities for preclinical studies focused on outcome improvements for congenital heart disease.

Single step production of Cas9 mRNA for zygote injection.

Production of Cas9 mRNA in vitro typically requires the addition of a 5´ cap and 3´ polyadenylation. A plasmid was constructed that harbored the T7 promoter followed by the EMCV IRES and a Cas9 coding region. We hypothesized that the use of the metastasis associated lung adenocarcinoma transcript 1 (Malat1) triplex structure downstream of an IRES/Cas9 expression cassette would make polyadenylation of in vitro produced mRNA unnecessary. A sequence from the mMalat1 gene was cloned downstream of the IRES/Cas9 cassette described above. An mRNA concentration curve was constructed with either commercially available Cas9 mRNA or the IRES/ Cas9/triplex, by injection into porcine zygotes. Blastocysts were genotyped to determine if differences existed in the percent of embryos modified. The concentration curve identified differences due to concentration and RNA type injected. Single step production of Cas9 mRNA provides an alternative source of Cas9 for use in zygote injections.

Quadrupling efficiency in production of genetically modified pigs through improved oocyte maturation.

Assisted reproductive technologies in all mammals are critically dependent on the quality of the oocytes used to produce embryos. For reasons not fully clear, oocytes matured in vitro tend to be much less competent to become fertilized, advance to the blastocyst stage, and give rise to live young than their in vivo-produced counterparts, particularly if they are derived from immature females. Here we show that a chemically defined maturation medium supplemented with three cytokines (FGF2, LIF, and IGF1) in combination, so-called "FLI medium," improves nuclear maturation of oocytes in cumulus-oocyte complexes derived from immature pig ovaries and provides a twofold increase in the efficiency of blastocyst production after in vitro fertilization. Transfer of such blastocysts to recipient females doubles mean litter size to about nine piglets per litter. Maturation of oocytes in FLI medium, therefore, effectively provides a fourfold increase in piglets born per oocyte collected. As they progress in culture, the FLI-matured cumulus-oocyte complexes display distinctly different kinetics of MAPK activation in the cumulus cells, much increased cumulus cell expansion, and an accelerated severance of cytoplasmic projections between the cumulus cells outside the zona pellucida and the oocyte within. These events likely underpin the improvement in oocyte quality achieved by using the FLI medium.

Glycine supplementation in vitro enhances porcine preimplantation embryo cell number and decreases apoptosis but does not lead to live births.

Most in vitro culture conditions are less-than-optimal for embryo development. Here, we used a transcriptional-profiling database to identify culture-induced differences in gene expression in porcine blastocysts compared to in vivo-produced counterparts. Genes involved in glycine transport (SLC6A9), glycine metabolism (GLDC, GCSH, DLD, and AMT), and serine metabolism (PSAT1, PSPH, and PHGDH) were differentially expressed. Addition of 10 mM glycine to the culture medium (currently containing 0.1 mM) reduced the abundance of SLC6A9 transcript and increased total cell number, primarily in the trophectoderm lineage (P = 0.003); this was likely by decreasing the percentage of apoptotic nuclei. As serine and glycine can be reversibly metabolized by serine hydroxymethyltransferase 2 (SHMT2), we assessed the abundance of SHMT2 transcript as well as its functional role by inhibiting it with aminomethylphosphonic acid (AMPA), a glycine analog, during in vitro culture. Both AMPA supplementation and elevated glycine decreased the mRNA abundance of SHMT2 and tumor protein p53 (TP53), which is activated in response to cellular stress, compared to controls (P ≤ 0.02). On the other hand, mitochondrial activity of blastocysts, mtDNA copy number, and abundance of mitochondria-related transcripts did not differ between control and 10 mM glycine culture conditions. Despite improvements to these metrics of blastocyst quality, transfer of embryos cultured in 10 mM glycine did not result in pregnancy whereas the transfer of in vitro-produced embryos cultured in control medium yielded live births. Mol. Reprod. Dev. 83: 246-258, 2016. © 2016 The Authors.

Meganucleases Revolutionize the Production of Genetically Engineered Pigs for the Study of Human Diseases.

Animal models of human diseases are critically necessary for developing an in-depth knowledge of disease development and progression. In addition, animal models are vital to the development of potential treatments or even cures for human diseases. Pigs are exceptional models as their size, physiology, and genetics are closer to that of humans than rodents. In this review, we discuss the use of pigs in human translational research and the evolving technology that has increased the efficiency of genetically engineering pigs. With the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated (Cas) protein 9 system technology, the cost and time it takes to genetically engineer pigs has markedly decreased. We will also discuss the use of another meganuclease, the transcription activator-like effector nucleases , to produce pigs with severe combined immunodeficiency by developing targeted modifications of the recombination activating gene 2 (RAG2).RAG2mutant pigs may become excellent animals to facilitate the development of xenotransplantation, regenerative medicine, and tumor biology. The use of pig biomedical models is vital for furthering the knowledge of, and for treating human, diseases.

PS48 can replace bovine serum albumin in pig embryo culture medium, and improve in vitro embryo development by phosphorylating AKT.

The application of embryo-related technology is dependent on in vitro culture systems. Unfortunately, most culture media are suboptimal and result in developmentally compromised embryos. Since embryo development is partially dependent upon Warburg Effect-like metabolism, our goal was to test the response of embryos treated with compounds that are known to stimulate or enhance this Effect. One such compound is 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48). When added during oocyte maturation, the quality of the resultant embryos was compromised, whereas when added to the culture medium after fertilization, PS48 improved both the percentage of embryos that reach the blastocyst stage and the number of nuclei in those blastocysts. Embryonic PS48 treatment resulted in more phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) in blastocyst-stage embryos as compared to the controls. Further, PS48 could replace bovine serum albumin in embryo culture medium, as demonstrated by high-quality embryos that were developmentally competent. The action of PS48 appears to be via stimulation of phosphoinositide-3 kinase and phosphorylation of AKT, which is consistent with stimulation of the Warburg Effect.

Genomic profiling to improve embryogenesis in the pig.

Over the past decade the technology to characterize transcription during embryogenesis has progressed from estimating a single transcript to a reliable description of the entire transcriptome. Northern blots were followed by sequencing ESTs, quantitative real time PCR, cDNA arrays, custom oligo arrays, and more recently, deep sequencing. The amount of information that can be generated is overwhelming. The challenge now is how to glean information from these vast data sets that can be used to understand development and to improve methods for creating and culturing embryos in vitro, and for reducing reproductive loss. The use of ESTs permitted the identification of SPP1 as an oviductal component that could reduce polyspermy. Microarrays identified LDL and NMDA as components to replace BSA in embryo culture media. Deep sequencing implicated arginine, glycine, and folate as components that should be adjusted in our current culture system, and identified a characteristic of embryo metabolism that is similar to cancer and stem cells. Not only will these characterizations aid in improving in vitro production of embryos, but will also be useful for identifying, or creating conditions for donor cells that will be more likely to result in normal development of cloned embryos. The easily found targets have been identified, and now more sophisticated methods are being employed to advance our understanding of embryogenesis. Here the technology to study the global transcriptome is reviewed followed by specific examples of how the technology has been used to understand and improve porcine embryogenesis both in vitro and in vivo.

Piglets produced from cloned blastocysts cultured in vitro with GM-CSF.

In general, pig embryos established by somatic cell nuclear transfer (SCNT) are transferred at the one-cell stage because of suboptimal embryo culture conditions. Improvements in embryo culture can increase the practical application of late embryo transfer. The goal of this study was to evaluate embryos cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) in vitro, and to track the in vivo developmental competency of SCNT-derived blastocysts from these GM-CSF embryos. The receptor for GM-CSF was up-regulated in in vitro-produced embryos when compared to in vivo-produced cohorts, but the level decreased when GM-CSF was present. In vitro fertilized (IVF) embryos, supplemented with GM-CSF (2 or 10 ng/ml), showed a higher frequency of development to the blastocyst stage compared to controls. The total cell numbers of the blastocysts also increased with supplementation of GM-CSF. Molecular analysis demonstrates that IVF-derived blastocysts cultured with GM-CSF exhibit less apoptotic activity. Similarly, an increase in development to the blastocyst stage and an increase in the average total-cell number in the blastocysts were observed when SCNT-derived embryos were cultured with either concentration of GM-CSF (2 or 10 ng/ml). When SCNT-derived embryos, cultured with 10 ng/ml GM-CSF, were transferred into six surrogates at Day 6, five of the surrogates became pregnant and delivered healthy piglets. Our findings suggest that supplementation of GM-CSF can provide better culture conditions for IVF- and SCNT-derived embryos, and pig SCNT-derived embryos cultured with GM-CSF in vitro can successfully produce piglets when transferred into surrogates at the blastocyst stage. Thus, it may be practical to begin performing SCNT-derived embryo transfer at the blastocyst stage.

Glycolysis in preimplantation development is partially controlled by the Warburg Effect.

Glucose metabolism in preimplantation embryos has traditionally been viewed from a somatic cell viewpoint. Here, we show that gene expression in early embryos is similar to rapidly dividing cancer cells. In vitro-produced pig blastocysts were subjected to deep-sequencing, and were found to express two gene variants that have been ascribed importance to cancer cell metabolism (HK2 and the M2 variant of PKM2). Development was monitored and gene expression was quantified in additional embryos cultured in low or high O(2) (5% CO(2), 5% O(2), 90% N(2) vs. 5% CO(2) in air). Development to the blastocyst stage in the two atmospheres was similar, except low O(2) resulted in more total and inner cell mass nuclei than high O(2). Of the 15 candidate genes selected that are involved in glucose metabolism, only TALDO1 and PDK1 were increased in the low O(2) environment. One paradigm that has been used to explain glycolysis under low oxygen tension is the Warburg Effect (WE). The WE predicts that expression of both HK2 and PKM2 M2 results in a slowing of glucose metabolism through the TCA cycle, thereby forcing the products of glycolysis to be metabolized through the pentose phosphate pathway and to lactic acid. This charging of the system is apparently so important to the early embryo that redundant mechanisms are present, that is, a fetal form of PKM2 and high levels of PDK1. Here, we set the framework for using the WE to describe glucose metabolism and energy production during preimplantation development.